Org Divers Evol (2010) 10:123–133 DOI 10.1007/s13127-010-0004-4 ORIGINAL ARTICLE Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus (Orthoptera: Acrididae: Gomphocerinae) Dirk Berger & Dragan P. Chobanov & Frieder Mayer Received: 2 September 2008 /Accepted: 15 April 2009 /Published online: 11 March 2010 # Gesellschaft für Biologische Systematik 2010 Abstract A warming climate leads to shifts in distribution between the two geographically separated populations of ranges to higher latitudes and altitudes. Consequently, cold- S. cotticus studied. We suppose that S. cotticus had a wider adapted alpine species can be trapped in interglacial distribution during colder periods, when its range was Holocene refugia on high mountain summits if they fail to expanded to lower altitudes. This hypothesis is supported expand their ranges to the north. One example is the alpine by the current distribution of the closely related montane S. grasshopper Stenobothrus cotticus. This species was rubicundulus. assumed to be endemic to the southwestern Alps (France, Italy). However, we have found a second refugium in the Keywords Distribution . Glacial expansion . Rila Mountains in southwestern Bulgaria. Analyses of the Interglacial refugia . Stenobothrus cotticus . mitochondrial gene co1 and of phenotypic characters from Stenobothrus rubicundulus morphology and behaviour did not reveal differences Introduction Electronic supplementary material The online version of this article (doi:10.1007/s13127-010-0004-4) contains supplementary material, which is available to authorized users. Major climatic fluctuations in about 100,000 year cycles are : well documented for the last 700,000 years when cold ice D. Berger F. Mayer ages (glacials) were separated by warm interglacials (Webb Institut für Biologie, Universität Erlangen-Nürnberg, Staudtstraße 5, and Bartlein 1992). Many investigations of plant and 91058 Erlangen, Germany animal species have shown concordantly that periodical changes in climate during the Quaternary have significantly D. P. Chobanov affected the distribution of species and led to substantial Institute of Zoology, Bulgarian Academy of Sciences, Tsar Osvoboditel blvd. 1, range shifts both in latitude and altitude (e.g. Comes and BG-1000 Sofia, Bulgaria Kadereit 1998; Hewitt 1996, 1999, 2004; Taberlet et al. 1998). The climatic changes among glacial-interglacial Present Address: cycles were not uniform. Warming (interstadials) and D. Berger (*) Senckenberg Naturhistorische Sammlungen Dresden, cooling periods (stadials) lasted from 70 years to several Museum für Tierkunde, millennia; annual mean temperatures could have changed Königsbrücker Landstraße 159, by 10–12°C in fewer than 10 years (Hewitt 1996; Müller et 01109 Dresden, Germany al. 2003). During the glacials the Arctic ice cap progres- e-mail: [email protected] sively expanded southwards. Animal and plant populations Present Address: followed their appropriate environments and moved to the F. Mayer south or to lower altitudes during cold periods. Therefore, Museum für Naturkunde, Leibniz-Institut für Evolutions- und thermophilic species became restricted to small refugia in Biodiversitätsforschung an der Humboldt-Universität zu Berlin, Invalidenstraße 43, the south, while their former ranges were occupied by cold- 10115 Berlin, Germany resistant species. During the interglacials the ice sheets 124 D. Berger et al. retreated northwards and to higher altitudes. Thermophilic help infer the historic biogeography of S. cotticus we species spread out from southern refugia and expanded studied the current distribution of its closest European their ranges northwards or to higher levels in the moun- relative, S. rubicundulus Kruseman & Jeekel, 1967, which tains. In contrast to many others, some cold adapted species is adapted to montane habitats. failed to expand their range to the north and became restricted to small mountain summits (‘sky islands’) that now serve as inter-glacial refugia (DeChaine and Martin Material and methods 2004; Knowles 2000, 2001). Grasshoppers of the subfamily Gomphocerinae have Material been studied intensively with the aim of understanding the impact of climatic changes on the distribution of species Bulgarian specimens of S. cotticus were collected at (e.g. Hewitt 1996, 1999). They are widely spread across the Razdela site (42°11′N23°19′E) above Sedemte Rilski Palaearctic and Nearctic and are adapted to a wide range of Ezera tourist hostel and on the surrounding Otovishki Vruh ecological settings. Species diversity is highest in the peak, Topilata range, Vurla peak, Damga peak and Dodov temperate climate zone, and the number of species Vruh peak (42°10′N23°20′E) between 2,300 m and decreases rapidly towards the dry as well as towards the 2,650 m above sea level (m). In order to compare the cold climate zones (Bei-Bienko and Mistshenko 1951). Bulgarian and French populations, a further excursion to Many species expanded their distribution range to the north Col d’Izoard in the French Hautes-Alpes (44°49′N06°44′E, after the last ice age (Cooper et al. 1995; Lunt et al. 1998) site elevation 2,307 m) was made. Furthermore, published and formed hybrid zones at the secondary contacts of distribution records were used to infer the distribution ranges populations that had been separated during glaciation of the closely related species S. cotticus and S. rubicundulus (Bridle and Butlin 2002; Butlin and Hewitt 1985; Hewitt (see the “Electronic Supplementary Material”). Additional 1975; Jiggins and Mallet 2000; Vedenina and von Helversen unpublished records were obtained by revising the collec- 2003). Hybridizing taxa often resemble each other in tions of the National Museum of Natural History, Sofia morphology but can be distinguished easily by their songs. (NMNH); Macedonian Museum of Natural History, Skopje Males generate songs by stridulatory movements of both hind (MMNH); Museum of Blagoevgrad (BHM); L. Stefanov legs rubbing against the forewings. Females respond acous- (Skopje); D. Chobanov (Sofia); D. Berger (Dresden); and O. tically to a singing male if specific song parameters match von Helversen (Erlangen). A distribution map was created their preferences. The songs and, hence, the underlying leg using the utilities of http://www.aquarius.geomar.de/. movement patterns evolve rapidly and can attain high levels The morphological features of ten males and four of complexity (Bull 1979;Elsner1968;vonHelversen1986; females from the Bulgarian population and 11 males and von Helversen and von Helversen 1994; Otte 1972). eight females from the French population were investigat- Therefore, stridulatory leg movement patterns and resulting ed. These included the two and three males, respectively, acoustic signals are highly informative phenotypic traits that of the Bulgarian and French populations for which the assist in the reconstruction of the evolutionary history of song was also recorded, as well as the two genetically populations and closely related species. investigated individuals. In the present study we investigated the biogeography and conservation status of an ecologically highly specialised Morphometric investigations alpine grasshopper, Stenobothrus cotticus Kruseman & Jeekel, 1967. This species was assigned to the subgenus Morphometric measurements were taken from dried grass- Crotalacris Chopard within the genus Stenobothrus Fischer hoppers using an ocular micrometer-equipped (Leitz Wetzlar (Harz 1975) on the basis of wing characters. The species of Germany, Periplan GF 10x) binocular (Zeiss-Jena Citoval Crotalacris show few morphological differences (Harz A10026). The following measurements were taken: Prono- 1975), but their songs are strikingly different (Berger tum length (measured along the median keel), tegmen length 2008;ElsnerandWasser1995a; Ragge and Reynolds (maximum length of the folded right fore wing), and length 1998), thus allow unequivocal identification of the of the right hind femur. Only intact parts of the animals were different species. Stenobothrus cotticus is currently found measured. only at highest altitudes and was assumed to be endemic to the southwestern Alps (Harz 1975;Voisin2003). Here Song analysis we describe a newly discovered refugium of S. cotticus on the Balkan Peninsula. We use morphological, behavioural Song recordings were made under controlled temperature and genetic traits to investigate the divergence between conditions (∼28°C) in the laboratory of the University the two geographically widely separated populations. To Erlangen-Nürnberg. We limited our acoustic analyses to Interglacial refugia and range shifts of the alpine grasshopper Stenobothrus cotticus 125 courtship songs, because these also contain the elements of Biotechnologie GmbH). The temperature cycle of 94°C for calling songs in S. cotticus and in S. rubicundulus (Berger 30 s, 50°C for 50 s, and 72°C for 90 s was repeated 35 2008; Ragge and Reynolds 1998; own unpublished data). A times. After PCR, two units exonuclease I (E. coli) and one female was placed next to a male to ensure recording of unit Antarctic phosphatase (both from New England courtship songs. Songs were recorded with a 1/2″ condenser BioLabs) in 5 µl H2O were added to the amplification microphone (G.R.A.S. Type 40AF) equipped with a G.R.A. products and incubated at 30°C for 15 min. Both enzymes S. 26AB preamplifier. The signal was amplified by a Brüel were denatured at 80°C for 15 min. & Kjær measuring amplifier (Type 2608). The sound-